Free Lysate Research Articles

Genomic characterization, in vitro, and preclinical evaluation of two microencapsulated lytic phages VB_ST_E15 and VB_ST_SPNIS2 against clinical multidrug-resistant Salmonella serovars

BackgroundSalmonella infections continue to be one of the essential public health issues threatening millions of people. With the increasing occurrence of resistance against conventionally used antibiotics, the search for alternatives has become crucial. In this study, we aimed to isolate, characterize, and evaluate two lytic bacteriophages against clinically isolated multidrug-resistant (MDR) Salmonella serovars.MethodsScreening for the phage lytic activity was performed using a spot test. Characterization of the isolated phages was done by determining the host range, longevity test, and the effect of temperature, pH, organic solvents, and morphological characterization using a transmission electron microscope. Genomic analysis was performed using Oxford nanopore sequencing. The lytic activities of the free phage lysates and formulated phage as microencapsulated were evaluated both in vitro and in vivo.ResultsTwo phages (VB_ST_E15 and VB_ST_SPNIS2) were successfully isolated and showed lytic strong activities against MDR Salmonella (S.) Typhimurium ATCC 14,028, S. Paratyphi A, and S. Typhi. The two phages survived at the tested temperatures, maintained their infectivity for 90 days, and retained their activity until 60 °C with thermal inactivation at 65 °C. They were lytic at a pH range from 3 to 11 but lost their activities at extremely acidic or alkaline pH. The phages could withstand the organic solvents but were completely inactivated by 100% ethanol. Both phages were classified under the order Caudoviricetes, and Genus: Uetakevirus. Their genomic sequences were assembled, annotated, and submitted to the NCBI GenBank database (OR757455 and OR757456). The preclinical evaluation using the murine animal model revealed that the two-phage cocktail managed MDR Salmonella infection as evidenced by the reduction in the bacterial burden, increased animal weight, and histopathological examination.ConclusionThe two encapsulated phage formulas could be considered promising candidates for the management of MDR Salmonella-associated infections and clinical analysis should be undertaken to evaluate their potential use in humans.

Spo0A-dependent antifungal activity of a plant growth promoting rhizobacteria Bacillus subtilis strain UD1022 against the dollar spot pathogen (Clarireedia jacksonii)

Dollar spot is an economically important foliar disease of turfgrass caused by Clarireedia spp. Increased use of chemical fungicides for disease management has possible negative effects on the environment and human health. The quest for eco-friendly alternatives to fungicides is driving the development of new biological strategies for managing dollar spot. In the present study, an analysis of antifungal activity of the plant growth promoting rhizobacteria (PGPR) Bacillus subtilis strain UD1022 (hereafter UD1022) and plant health products (PHPs) (Acibenzolar-S-methyl, kelp extract, fosetyl-Al, and trinexapac-ethyl) against Clarireedia jacksonii for control of the turfgrass fungal disease dollar spot was conducted. In vitro assays revealed that UD1022 has significant inhibitory effects against the mycelial growth of C. jacksonii. Of all the treatments, wild type UD1022 and a non-ribosomal peptide mutant (sfp¯) showed potential antagonism against C. jacksonii with inhibition rate of 17.2 % and 18.4%, respectively. Interestingly a Bacillus-derived broad antifungal compound (surfactin) and PHPs showed no direct antagonistic activity against dollar spot pathogen and were statistically like the untreated control. All the mutants of UD1022 except spo0A¯ showed varying but significant inhibition of the fungal pathogen suggesting that spo0A gene may be directly involved in the antagonism against C. jacksonii. Bacterial volatile compounds (BVCs) of UD1022 may not be involved in antagonism as no fungal inhibition was observed in the indirect assay using split petri plates. Filter sterilized cell free lysate (CFL) as well as heat treated CFL of UD1022 bacterial culture did not show any fungal inhibition indicating a primary role of live bacterial cells and not bacterial-derived or secreted compounds in fungal growth inhibition. The results indicate UD1022 to possess the potential to be extended for the biocontrol of dollar spot of turfgrass.

Abstract 1775: Expansion of human T cells using xenogeneic free and gamma irradiated human platelet lysate

Abstract Commercially available human platelet lysate (hPL) is produced using expired human platelets obtained from accredited blood banks in the United States. These platelets were originally intended for use in patient transfusion. The safety of platelets used in transfusion is managed by the U.S. Food & Drug Administration (FDA), as well as the American Association of Blood Banks (AABB). These organizations set standards, including testing for transmissible diseases. The United States record for blood safety is well established, with extremely low rates of disease transmission, making the platelet units used for hPL manufacture low risk. The Covid-19 pandemic has increased awareness of emerging infectious diseases, even though transmission of Covid-19 via blood transfusion has not been documented. For that reason, gamma irradiated hPL offers an additional safety measure in the clinic. Chimeric Antigen Receptor (CAR) expressing T-cells have demonstrated potent clinical efficacy in patients with hematological malignancies. In addition, there are several phase I clinical trials evaluating the use of CAR-T-cells for targeting of solid tumor-associated antigens. Some of the challenging issues found during production of CAR-T cells are the efficiency of T cell transduction to generate CAR-T cells, the expansion of T cells to clinically relevant numbers and the long-term survival in vivo of the therapeutic cells. The use of human platelet lysate has been demonstrated to improve these issues. Our data from experiments performed using human CD3+ from donors demonstrates that human platelet lysates offer an improved performance on T cell expansion versus serum derived products. hPL efficiently promotes T cell expansion, with higher cell yields and lower cell exhaustion rate. Additionally, we efficiently developed a protocol for suspension culture of T cells, which could facilitate the large-scale expansion of allogeneic CAR-T cells. Citation Format: Vanesa Alonso-Camino. Expansion of human T cells using xenogeneic free and gamma irradiated human platelet lysate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1775.

The main Aflatoxin B1 degrading enzyme in Pseudomonas putida is thermostable lipase

Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin mainly produced by Aspergillus flavus and A. parasiticus, and prevalent in food and feed. Microbial degradation is a promising strategy which can be performed in mild and environmental friendly condition. This work is a step towards identifying the enzyme responsible for biodegradation of AFB1 by P. putida. Experiments were performed with P. putida lysate and compared with commercial lipase to see the degradation efficiency and the temperature stability. The cell free lysate of P. putida efficiently degraded AFB1 in a range of temperature from 20 to 90 °C. The lysate is thermostable and could retain its activity on pre-incubation up to 90 °C. Highest rate of degradation was observed at 70 °C. These observations show that the P. putida lysate is not only stable at higher temperatures but its enzymatic activity increases after incubation. Similarly, the commercial lipase degraded AFB1 efficiently. However, both, the P. putida lysate and lipase ceased degradation in presence of a lipase inhibitor, HgCl2. The Hill function accurately predicted enzyme activity at various times and temperatures. Like lipase, the lysate also hydrolyses the p-nitrophenyl palmitate to p-nitrophenol. Kinetic parameters such as Vmax, Km and n values are good measures to characterize the lysate response with respect to changing paranitro phenyl palmitate levels. The substrate specificity test of lipase showed linear correlation between the absorbance at 410 nm vs amount of product paranitro phenol. The value of Km, Vmax and n are 0.62 mM, 355.7 μmol min−1 and 1.29, respectively. The lipase gene presence in P. putida was confirmed using PCR technique. These observations indicate that the main enzyme responsible for AFB1 degradation by P. putida is lipase. Thus, lipase as a multifunctional biocatalyst provides a promising future for a variety of industries and may also help to ensure the food safety by degrading the mycotoxins.

Polymer Nanoparticle-Mediated Delivery of Oxidized Tumor Lysate-Based Cancer Vaccines.

Cancer vaccination is a powerful strategy to combat cancer. A very attractive approach to prime the immune system against cancer cells involves the use of tumor lysate as antigen source. The immunogenicity of tumor lysate can be further enhanced by treatment with hypochlorous acid. This study explores poly(lactic-co-glycolic acid) (PLGA) nanoparticles to enhance the delivery of oxidized tumor lysate to dendritic cells. Using human donor-derived dendritic cells, it is found that the use of PLGA nanoparticles enhances antigen uptake and dendritic cell maturation, as compared to the use of the free tumor lysate. The ability of the activated dendritic cells to stimulate autologous peripheral blood mononuclear cells (PBMCs) is assessed in vitro by coculturing PBMCs with A375 melanoma cells. Live cell imaging analysis of this experiment highlights the potential of nanoparticle-mediated dendritic-cell-based vaccination approaches. Finally, the efficacy of the PLGA nanoparticle formulation is evaluated in vivo in a therapeutic vaccination study using B16F10 tumor-bearing C57BL/6J mice. Animals that are challenged with the polymer nanoparticle-based oxidized tumor lysate formulation survive for up to 50 days, in contrast to a maximum of 41 days for the group that receives the corresponding free oxidized tumor lysate-based vaccine.

Bioprocess development for efficient conversion of CO2 into calcium carbonate using keratin microparticles immobilized Corynebacterium flavescens

Conversion of CO2 into calcium carbonate using microorganism is one of the promising methods of reducing the increased level of CO2 in the atmosphere. In the present study, a novel strain of Corynebacterium flavescens was isolated from cow saliva and screened for CO2 hydration activity. First of all, the effect of C. flavescens on CaCO3 precipitation was studied under optimized conditions. Various factors such as CO2 concentration, protein concentration, pH of the reaction system, and temperature were then optimized for maximum CO2 precipitation into CaCO3 using free cell lysate. After that, C. flavescens cells were immobilized onto highly porous keratin microparticles functionalized with glutaraldehyde (0.6 % v/v) to enhance the CO2 conversion efficiency. Maximum immobilization of bacterial cells was achieved after 25 h at 4 °C under shaking conditions. The covalently immobilized C. flavescens particles showed a 1.35-fold increase in CaCO3 production compared to their free counterparts. Under repeated batch conditions, the relative production of CaCO3 for immobilized and free cells was 53.46 and 22.15 %, respectively, after 10 cycles. This study demonstrated the advantages of keratin microparticles as a support for the immobilization of C. flavescens and the efficient conversion of CO2 to CaCO3.

Cloning and Overexpression of Strictosidine β-D-Glucosidase Gene Short Sequence from Catharanthus roseus in Escherichia coli.

Purpose: Strictosidine-β-D-glucosidase (SGD) is considered as a key enzyme in the production of bisindole alkaloids in Catharanthus roseus. The present study illustrated the production of a short sequence of this enzyme in Escherichia coli without codon optimization. Methods: Strictosidine-β-D-glucosidase (sgd) gene short sequence (1434 bp), which lacks the conserved sequence KGFFVWS and the localization peptide sequence at the C-terminal, was amplified from cDNA of C. roseus leaves, cloned and expressed in Escherichia coli. The activity of the produced protein in cell free lysate was tested using total alkaloid extract of C. roseus leaves. Results: HPLC and LC-MS analysis of the assay mixture revealed the disappearance of the strictosidine peak. Conclusion: SGD short sequence can be produced in Escherichia coli in active form without codon optimization.

Quorum quenching activity of Bacillus cereus isolate 30b confers antipathogenic effects in Pseudomonas aeruginosa.

Background: Quorum quenching, the interference of a Quorum sensing (QS) system that contributes to the pathogenesis through triggering the production of various virulence determinants, is among the newly suggested antivirulence strategies.Purpose: This study aimed at screening of N-Acyl homoserine lactonase activity from local bacterial isolate, and investigating its effect on Pseudomonas aeruginosa (P. aeruginosa) virulence and biofilm formation.Materials and methods: Soil bacteria were screened for aiiA gene coding for lactonase enzyme by Polymerase Chain reaction and sequencing of aiiA gene homologs. Lactonase activity and spectrum were assessed in the cell-free lysate by well diffusion assay using Agrobacterium tumafaciens KYC55. A bacterial isolate showing the highest N-acyl-homoserine lactones degradation percentage was identified by gene amplification and sequencing of the 16S rRNA gene and its aiiA gene homolog. High performance liquid chromatography was used to confirm N-acyl-homoserine lactone degradation. The effect of cell-free lysate on the biofilm formation ability and cytotoxicity of P. aeruginosa PAO1 and P. aeruginosa clinical isolates from different clinical sources were assessed by static microtiter plate and viability assay, respectivelyResults: Lactonase gene and activity were identified in three Bacillus spp. isolates. They showed broad catalytic activities against tested N-acyl-homoserine lactones. However, The lactonase activity in the cell- free lysate of isolate 30b showed the highest significant degradation percentage on all tested signals; N-butanoyl-L-homoserine lactone (71%), N-hexanoyl-l-homoserine lactone (100%), N-decanoyl-homoserine lactone (100%), N-(3-oxohexanoyl)-L-homoserine lactone (37.5%), N-(oxodecanoyl)-L-homoserine lactone (100%), and N-(3-oxododecanoyl)-L-homoserine lactone (100%). Alignment of the amino acid sequences of AiiA protein of isolate 30b showed 96% identity with Bacillus cereus (B. cereus) homologous lactonases in the GenBank database, and the isolate was designated as B. cereus isolate 30b. Cell-free lysate of B. cereus isolate 30b reduced biofilm formation significantly in 93% of P. aeruginosa isolates. The highest mean percentage of reduction in the biofilm was 86%. Moreover, the viability percentage of human lung carcinoma A549 cells infected by P. aeruginosa and treated with cell-free lysate of B. cereus isolate 30b increased up to 15%.Conclusion: The results of this study highlight the potential of lactonases as a promising strategy to combat Pseudomonas aeruginosa virulence.

Abstract 4041: K-ras transformation leads to increased HS-degradation

Abstract INTRODUCTION: Degradation of glycosaminoglycans in the extracellular matrix impacts cell movement, angiogenesis and other cancer cell activities. The purpose of study was to detect and measure extracellular heparan sulfate (HS)-degrading enzyme in malignant ras-transformed cells where HS degradation occurred at attachment sites. METHODS: Serum free conditioned medium (CM) and lysate from subconfluent Balb/c3T3 and KiMSV (Ki-ras-transformed 3T3) cells were collected and protease inhibitors added. CM was concentrated by ultracentrifugal filters using 3K and 30K MWCO. Heparan degrading enzyme assay kit was use with lysate and CM for each cell line. Western blot analysis of lysate and CM used anti-HPSE, anti-NAP2, and anti-PPBP antibodies with actin control. mRNA for HPSE and PPBP was determined by RTPCR. RESULTS: The heparan degrading activity of CM from KiMSV cells was 12 fold higher than from Balb/c3T3 cells with 30K MWCO concentration. Further, HPSE was detected in the lysate, but not CM. Anti-NAP2 and anti-PPBP antibody detected protein greater than 30kD in CM and lysate for KiMSV cells. However, mRNA expression of PPBP/NAP2 was less in KiMSV than Balb/c3T3 cells. CONCLUSION: HS degradation in the CM of KiMSV cells is greater than for Balb/c3T3 cells from which they are derived after Ki-ras-transformation. The HPSE is not detected in the CM, but PPBP-like proteins at higher molecular weight are detected which may be responsible for the extracellular HS degradation. Citation Format: Donghong Ju, Mary A. Kosir. K-ras transformation leads to increased HS-degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4041.

Evaluation of Biotechnological Potential of Novel Mercury Tolerant Strain of Klebsiella Pneumonia

In order to investigate the effect of elevated mercury pollution on antibiotic tolerance and enzyme activities of human pathogens, we isolated a novel K. pneumoniae strain from the soil of a metallurgical dump yard that was capable of tolerating 250 μM HgCl2. Increasing concentration of Hg2+ in growth medium led to increased tolerance of the strain to ampicillin that was accompanied by increased secretion of extracellular polymeric substance (EPS). These results indicate that Hg2+-induced increased synthesis of EPS might be a key factor for increased tolerance of K. pneumoniae to ampicillin. The strain exhibited high catalase, alkaline phosphatase, mercuric reductase, amylase and lipase activity. Presence of 250 μM HgCl2 in growth medium increased mercuric reductase activity to 3 fold, however, no significant alteration in catalase, alkaline phosphatase and lipase was observed. The strain retained 86% amylase activity when grown in presence of 250 μM Hg2+. However, cell free lysate of the bacterium exhibited sensitivity towards Hg2+ leading to 30 to 40% decrease of these enzyme activities at 250 μM Hg2+. These results show that the bacterium possesses cellular mechanism to protect its enzymes from exogenous HgCl2. Our study reveals that increased heavy metal in the environment is a key factor leading to cross resistance of K. pneumoniae against membrane-targeted antibiotics ampicillin. Further, it’s a novel mercury-tolerant strain of K. pneumoniae isolate that exhibits high biotechnological potential for simultaneous bioremediation of mercury and production of industrially important enzymes.

Improvement of Bioconversion of Vitamin D3 into Calcitriol by Actinomyces hyovaginalis through Protoplast Fusion and Enzyme Immobilization

Protoplast fusion and enzyme immobilization techniques were applied to increase calcitriol production from vitamin D 3 using Actinomyces hyovaginalis , a local isolate recovered from Egyptian soil, that has a potential bioconversion activity of vitamin D 3 into calcitriol. A total of sixteen protoplast hybrids, formed between Actinomyces hyovaginalis isolate and two Bacillus species ( B. thuringiensis and B. weihenstephanensis ) were screened for vitamin D 3 bioconversion activity. Compared to wild type isolate, four hybrids (formed between Actinomyces hyovaginalis isolate and B. thuringiensis ) were found to preserve the bioconversion activity; out of which, three hybrids coded V2B, V3B and V8A exhibited higher calcitriol production. The hybrids coded V2B and V8A produced, per 1 L culture medium, about 0.5 and 0.4 mg calcitriol corresponding to 350% and 280%, respectively, increase compared to the wild type isolate. Among different alginate concentrations applied, immobilization of cell lysate of Actinomyces hyovaginalis isolate using 2% alginate showed 140% increase in calcitriol production from vitamin D 3 compared to the free cell lysate. Activity of the immobilized form was preserved for five repetitive uses over a period of 15 days but with a 50% decline in production occurring at the fifth use.

Canine CD4+CD8+ double-positive T cells participate in immunity to Ehrlichia chaffeensis infection

Abstract Ehrlichia chaffeensis is an obligate intracellular gram-negative bacteria and a frequent cause of severe and fatal tick-borne infection in people in North America. The reservoir host for E. chaffeensis is the white-tailed deer, while humans, dogs, coyotes and goats are regarded as common incidental hosts. In dogs, we have shown that E. chaffeensis establishes a chronic infection that persists for at least 42 days, while promoting development of Th1 and Th17 cellular responses and pathogen-specific humoral immunity. To date, little is known about the immune responses that correlate with control or successful clearance of the infection in dogs or humans. In dogs, a population of mature CD4+CD8+ double-positive (DP) T cells exist that have features of activated T cells and share some similarities to the CD4+CD8+ DP populations which have been described in pigs and humans. However, little is known about the function of these cells in dogs, particularly in the context of infectious diseases. Here, we demonstrate that canine CD4+CD8+ DP T cells expand significantly in the blood and spleen in response E. chaffeensis infection, and upregulate markers of T cell activation and antigen-experience. Recall responses after in vitro restimulation with E. chaffeensis host-cell free lysate showed that CD4+CD8+ DP T cells undergo multiple rounds of antigen-specific proliferation, produce IFNγ and IL-17, and upregulate expression of the cytotoxic molecules granzyme B and granulysin. Together, our results demonstrate that CD4+CD8+ DP T cells contribute to the immune response to E chaffeensis in the physiologic host, and suggest the importance of alternative lymphocyte populations in promoting protection and control of tick-borne diseases in the dog.

In Vitro Recombinant Expression of Novel Protective Protein Highly Accumulated in Dry State in an Anhydrobiotic Insect

Recent intensive studies showed that ability of several groups of organisms to withstand complete desiccation (anhydrobiosis state) is largely based on the activity of several groups of key proteins, including representatives of intrinsically disordered ones. In the largest known anhydrobiotic animal, the sleeping chironomid, both proteomic and genome-wide/targeted mRNA expression analysis suggest that several groups of the novel proteins greatly contribute to the formation of the molecular shield. Among them, 114 aa-long protein, dryporin. Being one of the most abundant in the desiccated larvae on protein level and showing one of the highest expression on mRNA level, this protein shows lack of the obvious homology with other known proteins. In this study, we successfully produced recombinant dryporin protein using BY-2 cell free lysate and showed the evidence of post-translational modification of the protein in the anhydrobiotic larvae.

Aflatoxin B1 degradation by liquid cultures and lysates of three bacterial strains

Aflatoxin contamination remains a daunting issue to address in food safety. In spite of the efforts geared towards prevention and elimination of this toxin, it still persists in agricultural commodities. This has necessitated the search for other measures such as microbial degradation to combat this hazard. In this study, we investigated the biodegradation of aflatoxin B1 (AFB1), using lysates of three bacterial strains (Pseudomonas anguilliseptica VGF1, Pseudomonas fluorescens and Staphylococcus sp. VGF2) isolated from a gold mine aquifer. The bacterial cells were intermittently lysed in the presence and absence of protease inhibitors to obtain protease free lysates, subsequently incubated with AFB1 for 3, 6, 12, 24, and 48h to investigate whether any possible AFB1 degradation occurred using high performance liquid chromatography (HPLC) for detection. Results obtained revealed that after 6h of incubation, protease inhibited lysates of Staphylococcus sp. VGF2 demonstrated the highest degradation capacity of 100%, whereas P. anguilliseptica VGF1 and P. fluorescens lysates degraded AFB1 by 66.5 and 63%, respectively. After further incubation to 12h, no residual AFB1 was detected for all the lysates. Lower degrading ability was however observed for liquid cultures and uninhibited lysates. Data on cytotoxicity studies against human lymphocytes showed that the degraded products were less toxic than the parent AFB1. From this study, it can thus be deduced that the mechanism of degradation by these bacterial lysates is enzymatic. This study shows the efficacy of crude bacterial lysates for detoxifying AFB1 indicating potential for application in the food and feed industry.

A high fat diet induces sex-specific differences in hepatic lipid metabolism and nitrite/nitrate in rats

Men and women differ substantially with regard to the severity of insulin resistance (IR) but the underlying mechanism(s) of how this occurs is poorly characterized. We investigated whether a high fat (HF) diet resulted in sex-specific differences in nitrite/nitrate production and lipid metabolism and whether these variances may contribute to altered obesity-induced IR.Male and female Wistar rats were fed a standard laboratory diet or a HF diet for 10 weeks. The level of plasma nitrite/nitrate, as well as free fatty acid (FFA), in both plasma and liver lysates were assessed. The levels of inducible nitric oxide (NO) synthase (iNOS), p65 subunit of NFκB, total and phosphorylated forms of Akt, mTOR and PDK-1 in lysates, and the levels of glucose transporter 2 (Glut-2) and fatty acid translocase/cluster of differentiation 36 (FAT/CD36) in plasma membrane fractions of liver were assessed.HF-fed male rats exhibited a significant increase in plasma nitrite/nitrate, and hepatic FFA and FAT/CD36 levels compared with controls. They also displayed a relative decrease in iNOS and Glut-2 levels in the liver. Phosphorylation of Akt (at Ser473 and Thr308), mTOR and PDK-1 was also reduced. HF-fed female rats exhibited increased levels of NFκB-p65 in liver compared with controls, while levels of Glut-2, FAT/CD36 and Akt phosphorylation at Thr308 and PDK-1 were decreased.Our results reveal that altered lipid and glucose metabolism in obesity, lead to altered iNOS expression and nitrite/nitrate production. It is likely that this mechanism contributes to sex-specific differences in the development of IR.

Invitro translation in a hybrid cell free lysate with exogenous cellular ribosomes.

Cell free protein synthesis systems (CFPS) have been widely used to express proteins and to explore the pathways of gene expression. In the present manuscript, we describe the design of a novel adaptable hybrid invitro translation system which is assembled with ribosomes isolated from many different origins. We first show that this hybrid system exhibits all important features such as efficiency, sensitivity, reproducibility and the ability to translate specialized mRNAs in less than 1h. In addition, the unique design of this cell free assay makes it highly adaptable to utilize ribosomes isolated from many different organs, tissues or cell types.

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

The internal ribosome entry site (IRES) element located at the 5´untranslated genomic region of various RNA viruses mediates cap-independent initiation of translation. Picornavirus IRES activity is highly dependent on both its structural organization and its interaction with host factors. Small molecules able to interfere with RNA function are valuable candidates for antiviral agents. Here we show that a small molecule based on benzimidazole (IRAB) inhibited foot-and-mouth disease virus (FMDV) IRES-dependent protein synthesis in cells transfected with infectious RNA leading to a decrease of the virus titer, which was higher than that induced by a structurally related benzimidazole derivative. Interestingly, IRAB preferentially inhibited IRES-dependent translation in cell free systems in a dose-dependent manner. RNA structural analysis by SHAPE demonstrated an increased local flexibility of the IRES structure upon incubation with IRAB, which affected 3 stem-loops (SL) of domain 3. Fluorescence binding assays conducted with individual aminopurine-labeled oligoribonucleotides indicated that the SL3A binds IRAB (EC50 18 μM). Taken together, the results derived from SHAPE reactivity and fluorescence binding assays suggested that the target site of IRAB within the FMDV IRES might be a folded RNA structure that involves the entire apical region of domain 3. Our data suggest that the conformational changes induced by this compound on a specific region of the IRES structure which is essential for its activity is, at least in part, responsible for the reduced IRES efficiency observed in cell free lysates and, particularly, in RNA-transfected cells.

A therapeutic microparticle-based tumor lysate vaccine reduces spontaneous metastases in murine breast cancer.

Metastatic breast cancer is currently incurable, and available therapies are associated with severe toxicities. Induction of protective anti-tumor immunity is a promising therapeutic approach for disseminated breast cancer, as immune responses are (i) systemic; (ii) antigen-specific; and (iii) capable of generating long-lived "memory" populations that protect against future tumor recurrences. Pursuant with this approach, we have developed a novel heterologous prime/boost vaccination regimen that reduces spontaneous lung metastases in mice with established murine 4T1 adenocarcinoma breast tumors. In our studies, mice were orthotopically challenged with luciferase-expressing 4T1 tumor cells; luciferase expression was retained in vivo, enabling us to quantitatively track metastatic tumor growth via bioluminescent imaging. On day 6 post-challenge, mice received a therapeutic "prime" consisting of bulk tumor lysates encapsulated in poly(lactic-co-glycolic) acid (PLGA) microparticles (MPs). On day 11, mice received a "boost" composed of free tumor lysates plus a cocktail of Toll-like receptor (TLR)-stimulating adjuvants. Tumor progression was monitored in vaccinated and untreated mice for 25 days, a time at which 100% of untreated mice had detectable lung tumors. PLGA MPs injected subcutaneously trafficked to draining lymph nodes and were efficiently phagocytosed by dendritic cells (DCs) within 48 h. Our combination therapy reduced metastatic lung tumor burdens by 42% and did not induce autoimmunity. These findings illustrate that vaccines based upon MP delivery of tumor lysates can form the basis of an effective treatment for metastatic breast cancer and suggest that similar approaches may be both efficacious and well-tolerated in the clinic.

Neutralizing the detrimental effect of glutathione on precious metal catalysts.

We report our efforts to enable transition-metal catalysis in the presence of cellular debris, notably Escherichia coli cell free extracts and cell lysates. This challenging goal is hampered by the presence of thiols, mainly present in the form of glutathione (GSH), which poison precious metal catalysts. To overcome this, we evaluated a selection of oxidizing agents and electrophiles toward their potential to neutralize the detrimental effect of GSH on a Ir-based transfer hydrogenation catalyst. While the bare catalyst was severely inhibited by cellular debris, embedding the organometallic moiety within a host protein led to promising results in the presence of some neutralizing agents. In view of its complementary to natural enzymes, the asymmetric imine reductase based on the incorporation of a biotinylated iridium pianostool complex within streptavidin (Sav) isoforms was selected as a model reaction. Compared to purified protein samples, we show that pretreatment of cell free extracts and cell lysates containing Sav mutants with diamide affords up to >100 TON's and only a modest erosion of enantioselectivity.

Assessment of intracellular methotrexate and methotrexate‐polyglutamate metabolite concentrations in erythrocytes by ultrafast matrix‐assisted laser desorption/ionization triple quadrupole tandem mass spectrometry

A new ultrafast quantitative and high-throughput mass spectrometric method using matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry has been developed and validated for determination of intracellular erythrocyte concentrations of the antifolate drug methotrexate (MTX) and its polyglutamate metabolites. The method consists of a solid-phase extraction of MTX and MTX-polyglutamate metabolites from deproteinized erythrocyte lysates spiked with aminopterin as internal standard. The newly developed method was validated according to the most recent FDA guidelines on linearity, recovery, within-run and between-run accuracy and precision and stability of the analytes. The low limit of quantification (LLOQ) was 10 nmol/L for all analytes while the limit of detection (LOD) determined at a signal-to-noise (S/N) ratio = 3:1 in drug- free erythrocyte lysate was on average 0.3 nmol/L. After validation, the new method was used in the measurement of intracellular erythrocyte concentrations of MTX and MTX-polyglutamate metabolites (MTXPG2 to MTXPG7) in packed human erythrocyte samples collected from patients with rheumatoid arthritis receiving low-dose oral methotrexate therapy. Mean (SD) intracellular erythrocyte concentrations observed in patient samples were 12.8 (12.6), 12.4 (9.4), 44.4 (30.0), 33.6 (35.9) and 9.4 (8.2) nmol/L for MTX to MTXPG5, respectively, in 10(6) erythrocytes. The highest observed glutamylation degree of MTX was MTXPG5, the very long chain MTX-polyglutamate metabolites MTXPG6 and MTXPG7 were not detected in the packed erythrocyte pellets collected from rheumatoid arthritis patients.